This application focuses on the biology of blood coagulation factor X (F.X), and builds on work carried out in the previous funding cycles of this grant. F.X occupies a central role in coagulation, since it can be activated either through the extrinsic pathway (Vlla/tissue factor), or through the intrinsic pathway (IXa/VIIla). In earlier work, we had delineated a number of naturally occurring mutations in the F.X gene, and had carried out structure-function studies on the mutant proteins. More recently, we developed an in vitro expression system for production of recombinant F.X variants. Finally, using homologous recombination techniques, we have generated a F.X-deficient mouse that is characterized by mid-embryonic lethality of uncertain etiology in about half of the affected mice, and neonatal death from bleeding complications in the remainder. The mid-embryonic lethality is of interest in light of recent studies suggesting that F.X and its activated form F.Xa may have a plethora of biological functions unrelated to clot formation, including mitogenic activity, promotion of cytokine release and up-regulation of transcription factors. Consistent with these reports is our observation that FX, in contrast to other vitamin K-dependent clotting factors, is expressed in a variety of tissues in adult humans and in the developing mouse embryo. The objective of the first aim is to evaluate the pathophysiology of the mid-embryonic lethality, and determine the structural, functional and tissue-specific characteristics of a F.X molecule capable of rescuing the mid-embryonic lethality. Specifically, we will determine the sites and level of expression required and will also determine whether an active site is required (since some F.X-mediated signaling events an active site is not required). The goal of aims 2 and 3 is to develop a better understanding of the relative roles of the intrinsic and extnnsic pathways of blood coagulation, using both recombinant proteins and mice transgenic for variant FX species. We and others have reported the existence of FX variants with markedly asymmetric activation in the intrinsic (IXa/VIIla) and extrinsic (Vlla-TF) pathways. The existence of these variants is consistent with current data suggesting that macromolecular substrate recognition by the extrinsic and intrinsic tenases is determined to a large extent by contact surfaces distinct from the scissile bond and different for the intrinsic versus the extrinsic tenase. Blocking of these exosites may represent a novel approach to anticoagulation. We intend to synthesize these asymmetrically activated variants, characterize them in purified component assays, and test their in vivo effects by creating mice carrying these variants that can be activated through one or the other pathway but not through both. We will characterize both baseline hemostasis and response to prothrombotic stimuli in these animals. This application represents a synthesis of two longstanding areas of investigation in this laboratory.